The long-term objectives of this research training plan are to serve as a training mechanism to develop and hone the critical skills necessary for conducting translational research as a neuroscientist for Dr. Dayna R. Loyd. Using a postdoctoral research fellowship and a structured Masters in Clinical Investigation (MSCI) program as a formal training vehicle, Dr. Loyd will conduct a critical research project utilizing both basic science and clinical research methodologies to answer important questions on the peripheral mechanisms of craniofacial pain. The overall approach of the present F32 application is to characterize the distribution of serotonin (5HT) receptors and overall integrated actions of 5HT on rat and human trigeminal capsaicin- sensitive nociceptors (craniofacial pain transmitting sensory neurons). The transient receptor potential V1 channel (TRPV1), gaited by heat and capsaicin (the pungent ingredient in chili peppers), is highly expressed on trigeminal nociceptors and is critical to the development of hypersensitivity to pain. 5HT may increase the activity of TRPV1 as a mechanism to evoke pain via trigeminal nociceptors. Experiments outlined in this proposal aim to test the hypothesis that 5HT, acting at peripheral 5HT receptors, increases the activity of capsaicin-sensitive trigeminal sensory neurons. Specific Aim 1 will characterize the expression of 5HT receptors, utilizing a combination of florescent in situ hybridization and immunohistochemistry, in TRPV1-expressing trigeminal sensory neurons from rats with orofacial inflammation. Specific Aim 2 will determine whether 5HT activates and/or sensitizes rat capsaicin-sensitive sensory neurons in primary culture using cell signaling (intracellular calcium accumulation) and functional (proinflammatory peptide release) assays. Specific Aim 3 will analyze 5HT receptor expression on human TRPV1-expressing nociceptors in normal versus inflamed dental pulp using fluorescent immunohistochemistry. In addition, aim 3 will examine the ability of 5HT to increase proinflammatory peptide release from isolated capsaicin-sensitive human trigeminal nociceptors in vitro. The results of the proposed research are critical to understanding how peripheral serotonin is involved in modulating both rat and human trigeminal nociceptors. Serotonin has long been recognized as a pro- inflammatory mediator released at the site of injury by platelets, endothelial cells, and immune cells, however, the mechanism(s) by which serotonin acts on sensory neurons to modulate pain are unknown. This knowledge will provide insight on peripheral craniofacial pain mechanisms, which may uncover novel therapeutic targets previously unknown to science and medicine. Our preliminary data provide strong support for the hypothesis and the results will have a significant translational impact. PUBLIC HEALTH RELEVANCE: Craniofacial pain, such as migraine, represents a prevalent and expensive healthcare problem. This project will evaluate the potential modulation of rat and human trigeminal sensory neurons by serotonin. If so, then novel drugs targeting peripheral serotonin receptors may be highly effective at treating craniofacial pain.